PROJECT SUMMARY/ABSTRACT The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying structures from injury, in part, through the elaboration of the limbal and corneal epithelia. It is well- accepted that the limbal epithelium is the site of the corneal epithelial stem cells, which are crucial for maintaining the corneal epithelium; however, major questions remain unresolved concerning how the limbal epithelium is regulated. microRNAs (miRNAs) are a major class of regulatory molecules that are part of the RNAi silencing machinery. We demonstrated that miR-184 was the most abundant corneal epithelial miRNA, and that miR-184 had marked angiostatic properties, which makes excellent biological sense as it is vital that the cornea maintains avascularity. Surprisingly, given the prominence of miR-184 in limbal/corneal epithelial biology, little is known about its regulation. Our recent evidence suggests that miR-184 is negatively regulated by Ephrin-A3 (EFNA3), a member of the Eph/ephrin receptor tyrosine kinase family. EFNA3, is primarily restricted to limbal epithelial basal cells. Interestingly, overexpression of EFNA3 significantly reduced miR-184 levels. Furthermore, another corneal-preferred miRNA, miR-210, targets EFNA3, which could indirectly maintain miR-184 levels in the corneal epithelium. The lack of miR-210 in the limbal epithelium likely accounts for limbal epithelial EFNA3 expression. Our hypothesis is that a EFNA3/miR-210 axis negatively regulates miR-184, which enables proper limbal vascularity, an essential component of the stem cell niche; a relatively understudied area. To test this hypothesis, we will modulate miRNA and target protein levels in submerged cultures of human limbal and corneal epithelial cells and human microvascular endothelial cells. We will assess the functional consequences of such miRNA and protein modulations with a combination of biochemical, molecular biological, cell biological and physiological approaches. Another goal of this proposal is to realize the unlimited potential of miRNAs as therapeutic interventions to affect diseased tissues. Preliminary data indicates that a novel high density lipoprotein (HDL)-nanoparticle (NP) can deliver functional miRNAs into human corneal epithelial cells. Excitingly, when a Cy-3-taged HDL-NP solution was applied topically to resting mouse corneas, the Cy-3-tagged HDL-NPs were detected in the cytoplasm of corneal epithelial basal and wing cells. We propose to focus on how miR-HDL-NPs affect the biology of limbal/corneal epithelia in vivo. To accomplish this, we will use diabetic mice as a model of compromised corneal epithelial wound healing and topically treat with a HDL-NP conjugated miR-205, a pro-migration miRNA. We will also use mice lacking angiotensin converting enzyme 2 (ACE2) as a model of chronic corneal inflammation (e.g., bacterial keratitis or dry eye) and topically treat with a HDL-NP conjugated miR-146a, an anti-inflammatory miRNA. Ultimately our studies will provide a foundation for delivery of: (i) inhibitors of specific miRNAs or their targets; or (ii) miRNAs to patients with diseases that affect the ocular anterior epithelia.